IntroductionThe transcription factor GATA3 is involved in mammary gland development and is crucial for the maintenance of the differentiated status of luminal epithelial cells. The role of GATA3 in breast cancer as a tumor suppressor has been established, although insights into the mechanism of GATA3 expression loss are still required.

MethodsChromatin immunoprecipitation assays were conducted to study progestin modulation of recruitment of transcription factors to GATA3 promoter. We performed western blot and reverse RT-qPCR experiments to explore progestin regulation of GATA3 protein and mRNA expression respectively. Confocal microscopy and in vitro phosphorylation studies were conducted to examine progestin capacity to induce GATA3 serine phosphorylation in its 308 residue. GATA3 participation in progestin-induced breast cancer growth was addressed in in vitro proliferation and in vivo tumor growth experiments.

ResultsIn this study, we demonstrate that progestin-activated progesterone receptor PR reduces GATA3 expression through regulation at the transcriptional and post-translational levels in breast cancer cells. In the former mechanism, the histone methyltransferase enhancer of zeste homolog 2 is co-recruited with activated PR to a putative progesterone response element in the GATA3 proximal promoter, increasing H3K27me3 levels and inducing chromatin compaction, resulting in decreased GATA3 mRNA levels. This transcriptional regulation is coupled with increased GATA3 protein turnover through progestin-induced GATA3 phosphorylation at serine 308 followed by 26S proteasome-mediated degradation. Both molecular mechanisms converge to accomplish decreased GATA3 expression levels in breast cancer cells upon PR activation. In addition, we demonstrated that decreased GATA3 levels are required for progestin-induced upregulation of cyclin A2, which mediates the G1 to S phase transition of the cell cycle and was reported to be associated with poor prognosis in breast cancer. Finally, we showed that downregulation of GATA3 is required for progestin stimulation of both in vitro cell proliferation and in vivo tumor growth.

ConclusionsIn the present study, we reveal that progestin-induced PR activation leads to loss of GATA3 expression in breast cancer cells through transcriptional and post-translational regulation. Importantly, we demonstrate that GATA3 downregulation is required for progestin-induced upregulation of cyclin A2 and for progestin-induced in vitro and in vivo breast cancer cell growth.

AbbreviationsActDactinomycin D

ANOVAanalysis of variance

bpbase pair

Btzbortezomib

chFCScharcoal-stripped FCS

ChIPchromatin immunoprecipitation

CHXcycloheximide

DMEMDulbecco’s modified Eagle’s medium

ERestrogen receptor

EZH2enhancer of zeste homolog 2

FCSfetal calf serum

GFPgreen fluorescent protein

IHCimmunohistochemistry

MFImean fluorescence intensity

MMTVmouse mammary tumor virus

MPAmedroxyprogesterone acetate

PBpermeabilization buffer

PBSphosphate-buffered saline

PcGpolycomb group G

PKAprotein kinase

PKIprotein kinase inhibitor

PRprogesterone receptor

PREprogesterone response element

RT-qPCRreverse transcriptase quantitative polymerase chain reaction

siRNAsmall interfering RNA

TSStranslational start site

WTwild type

Electronic supplementary materialThe online version of this article doi:10.1186-s13058-014-0491-x contains supplementary material, which is available to authorized users.

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